An aging liver loses the ability to proliferate after partial hepatic resections leading to a higher mortality in the elderly. Our laboratory' investigates molecular mechanisms that control liver proliferation. Liver specific protein, CCAAT/Enhancer Binding Protein alpha (C/EBPalpha), is a strong inhibitor of liver proliferation. C/EBPalpha inhibits proliferation of young livers through direct interactions with cyclin dependent kinases 2 and 4. In adipose tissues, C/EBPalpha causes growth arrest via repression of E2F transcription. We have recently found that aging switches C/EBPalpha in liver from inhibition of cdks to repression of E2F, the pathway that normally operates in adipose tissues. In old livers, C/EBPalpha is observed in a high MW complex C/EBPalpha-Rb-E2F4-Brm. This complex occupies E2F-dependent promoters and blocks activation of genes whose expression is required for liver proliferation. A failure to diminish this complex after partial hepatectomy seems to be a major cause for the reduced proliferative response in old livers. Three components of the complex, Rb, C/EBPalpha and E2F4, are differentially phosphorylated in old livers. Phosphorylation of C/EBPalpha at Serl93 is a key event in the formation of the age-specific complex. We have recently identified cdk4 and PP2A as enzymes that regulate phosphorylation-dephosphorylation of Serl93 of C/EBPalpha. In this application, we propose to examine the effects of aging on these signal transduction pathways and determine their roles -in the appearance of the C/EBPalpha-Rb-E2F4-Brm complex and in the reduction of the proliferative response. Our working hypotheses are that: 1) Phosphorylation of C/EBPalpha, E2F4, and Rb promotes the formation of the age related complex, 2) Phosphorylation of Serl93 of C/EBPalpha in the liver is mediated by cdk4 and is reversed by the action of the PI3K-Akt-PP2A pathway, 3) Aging liver increases the age-specific complex by diminishing the PI3K-Akt-PP2A pathway and by activation of cdk4. In Specific Aim 1, we will examine mechanisms by which aging activates cdk4 and down-regulates activity of PP2A. Specific Aim 2 examines whether phosphorylation of Rb and E2F4 affects their ability to form the age-specific C/EBPalpha complex. In Specific Aim 3, additional proteins of the complex will be identified and their role in liver proliferation will be examined. Based on data obtained in this project, we are planning to start developing a strategy to correct liver proliferation in elderly.